Media solutions and methods for cryopreservation and thawing of in vitro fertilization specimens

ABSTRACT

A medium solution which will increase the growth, survival and ultimately the live birth rate of oocytes and embryos which have been or will be subjected to cryopreservation. The solution contains varied amounts of glucose, pyruvates, amino acids, vitamins K5 and C, antioxidants, fatty acids to supply the specimens with the chemical ingredients and uptake requirements required to recover and prosper during and after the cryopreservation process. The solution supplies nutrients to the specimens that will replenish depletion and damage to the specimens and their mitochondria, spindles and structural features, such as cell walls. One formulation addresses the additional requirements of frozen specimens as opposed to the current media solutions and methods which treat the un-frozen specimens the same as the frozen specimens when recovering them from cryopreservation.

This application is a division of copending U.S. Ser. No. 12/454,942,filed May 27, 2009 which claims the benefit of Provisional PatentApplication Ser. No. 61/130,058, filed May 28, 2008.

FIELD OF THE INVENTION

Media solutions and methods of using the same for the restoration,resuscitation and treatment of oocytes, embryos and stem cells beforeand after cryopreservation or vitrification.

BACKGROUND OF THE RELATED ART

Human and other animal cells are presently being frozen, stored, andthen thawed as a means of saving these materials for use at a laterdate. Currently, human embryos are frozen and/or vitrified to accomplishcryopreservation. The terms frozen, vitrification and cryopreservationare interchangeable for the purpose of the subject disclosure.

The problem facing current methods of cryopreservation is the recoveringprocess and then the culturing to implantation for oocyte and embryos,and the resulting actual live birth. Currently, the freezing and thawingof a human oocyte may result in approximately a 10% overall live birthrate, which is well below the actual live birth rate of non-frozenoocyte or embryos. The conditions affecting the oocyte also affectembryos which are frozen and thawed and then implanted. In other words,there is a reduced rate of live birth for the frozen embryos as comparedto the non-frozen embryos.

The prior methods for the preparation and cryopreservation of specimenssuch as embryos, oocyte and stem cells are centered on separate anddifferent solutions and chemical ingredients.

When the time comes to thaw the specimens the current methods utilize aprogression of media that contain a combination of solutions used insequence. Again one may use up to seven different media solutions thatare in separate vials. The more common methods are a progression ofmedia reducing the level of sucrose in each and then the last solutionwill be sucrose free and used for washing. After a specimen is thawed itis transfered into a generic solution or off the shelf culture mediasolution used for the normal culturing of oocyte and embryos and do notaddress any changes in concentrations or the addition of new ingredientsto compensate for the freezing and thawing process.

In the current methods of cryopreservation, the specimen is immersedinto a series of media and cryoprotectorants whereby the concentrationof the cryoprotectorants (CP) is increased until, in some methods, theCP is 50% of the solution. The resulting infusion of the CP into thecells displaces water in the cells and dehydrates the cells during thisintroduction of the CP. This can be harmful to the cells. The cells arethen vitrified and then plunged into liquid nitrogen (LN) at atemperature as low as −180 C. The specimens will then be stored in theLN until they are to be used.

The specimens, when needed, are removed from the LN and then immersed ina series of media used to thaw the specimens. Currently, most of thesemedia contain a solution consisting of a base saline medium, HSA (10-20%of solution) and sucrose as an energy source. In one example Vial #1will contain 0.85 mM of sucrose. Vials #2 through #4 will decrease thesucrose concentrations stepwise down to 0.10 mM in Vial #4, and thenVial #5 will contain only a base media and HSA, and no sucrose.

The specimens will then be placed into a “generic” culture media untilusage. Embryos will be held in this medium until they are transferedinto the patient. Currently, embryos used in human IVF will be immersedin several media, embryo culture media, such as P1, HTF, HTF w/SSS andIrvine ECM® (Irvine Scientific), G2.2 through G5 series (Vitrolife),Quinn's Advantage (Sage), Cook® Cleavage and Cook® Blastocyst (CookIVF), Universal IVF medium®, EmbryoAssist™, BlastAssist™, ISM1 and 2™amd others (MediCult), Sigma Ham's F-10 and MEM 199 (Sigma), and Global®(LifeGlobal). These media have not been modified to accommodate theproblems being faced by the specimens in their post thawing development.They are not modified to be different for nonfrozen embryos asdifferentiated from frozen and then thawed embryos.

The introduction of the CP in the freezing process often results inproblems with and damage to the cells, such as dehydration, membranedamage, damage to the spindle, DNA damage, alterations to proteins,stripping of proteins, may alter ingredients, as well as otherstructural and physical changes and damage. For example, the frozenstate can result in damage to the cell walls and to the mitochondria.

The freezing process may introduce other factors such as ice formationduring either cooling or warming, and mechanical disruption of cellscontributing to the destabilization of membranes. Severe dehydrationfrom the exposure to concentrated CP can cause the structuraltransitions of biological molecules including lipids and proteins.

It appears that oocytes and embryos may lose or have certain diminishedmetabolic levels within the oocytes or embryos after the currentfreezing and thawing procedures, and the current practices do notaddress these freeze related extractions of certain proteins ornutrients when the CP is extracted or added.

The problems facing the specimens is that they may not recover from thefreeze, may not be fully flushed of the cryoprotectorant, may havesuffered physical damage to membranes or mitochondria and may not beprovided with the correct dosage of the required nutrients oringredients in order to recover. The problem facing the specimens isthat most media used in the freezing and thawing processes are those“generic” media which are used for the culture and development ofnonfrozen specimens, and which have different requirements.

In the current methods used for cryopreservation techniques, thespecimens are not “cultured” or “aided” in the process when they aregoing to be placed into cryopreservation or thawed out ofcryopreservation. The users utilize “generic” media, as they do in postthawing, as mentioned earlier, and do not add or alter any ingredientswhich may better prepare the specimen for cryopreservation, thawing orrecovery.

The specimens are placed into vitrification kits, which do not addresspre-vitrification or post vitrification conditions. Currently, embryosused in human IVF will be immersed in several vitrification solutions aspart of a sequential kit, such as Embryo and Blastocyst Freeze and ThawKit, Blastocyst Vitrification Freeze and Thaw and others (IrvineScientific), G-FreezeKit Blast™, RapidVit™ Cleave, RapidWarm™ Cleave andothers (Vitrolife), Quinn's Advantage® embryo freeze kit, Quinn'sAdvantage® blastocyst freeze kit (Sage), Blastocyst Cryopreservation™and Blastocyst Vitrification™ (Cook IVF), OocyteFreeze™ and Thaw™,BlastFreeze™ and BlastThaw™ and others (MediCult). These vitrificationsolutions, used in sequence do not contain a solution or method ofimmersing the embryos in a specially prepared, modified media toaccommodate the problems being faced by the specimens of theirpre-vitrification or post thawing procedures. They do not modify themedia, solutions or methods so as to be different for nonfrozen embryosas differentiated from frozen, and then thawed, embryos.

SUMMARY OF THE INVENTION

The subject invention relates to methods and culture media which willassist and enhance the rehabilitation, recovery and growth of specimenssuch as embryos, oocytes and stem cells after they have been subjectedto a vitrification or freezing process, and subsequently thawed, andwill provide important enhancements and ingredients to the specimens,which will allow them to recover, rebuild, and grow to the implantationstage and, in the case of embryos, result in increased live births. Thesubject invention helps to correct changes and deficiencies createdduring the preparation for freezing, the actual freezing process anddeficiencies which will be mitigated during the thawing or warming ofthe specimens.

The media and methods of this invention better provides the specimenswith necessary ingredients at various levels of concentration to giveeach specimen a better chance for recovery, survival, rebuilding andultimately live births in the case of embryo specimens. The subjectinvention addresses changes, the depletion of certain chemicals andstructural alterations, and damage resulting from the cryopreservationprocess.

The adaptation of a medium to become a recovery medium will require thealtering or adding of certain ingredients such as glucose, pyruvate andlactate, fatty acids, such as lipoic and linoleic acids, antioxidants,such as alpha tocopherol acetate (Vit. E) and ascorbic acid (Vit. C).Vitamins such as C and K, may contain cysteamine and chelators, such asEDTA, alpha and beta globulins and gamma globulin. Therefore most othermedia may already exceed the amount of glucose to be used and do nothave the same ratios as this invention's composition has, after thechanges. The problem facing most common media is their inability toestablish the same ratios of glucose, lactate and pyruvate, fatty acids,antioxidants, vitamins and alpha and beta globulins and gamma globulin,without affecting the remaining ingredients. Vitamin K5 is one of the Kvitamins which is suitable for use in the medium of this invention.

The adaptation of the media to be used inside or outside the incubatorwould greatly benefit the specimens. By including or not including HEPESor MOPS, bicarbonate buffers, would give these special media solutionsthe ability to be used in both the thawing process and culturingprocess. Making the special media solutions available, without HEPES,allows the extended culturing of the specimens, and by adding HEPES orMOPS allows the media to be used outside the incubator as most of thethawing protocols call for.

It would be highly desirable to provide media solution that wouldprovide thawing and post thawing embryos, oocytes and stem cells withingredients that will aid in the recovery process and to repairconditions that are a result of cryopreservation preparation, freezingand thawing.

It would be highly desirable to provide a media that replenishes energysources, proteins and repair membrane damage of the specimen which werealtered, stripped or damaged during the CP process.

It would be highly desirable to provide a media solution which will aidin the recovery and repair process of cells, oocyte, embryos and stemcells and result in a higher recovery and useful rate and in the case ofoocyte and embryos increase the live birth rate.

It should be appreciated that the present invention can be implementedand utilized in numerous ways, including without limitation as aprocess, an apparatus, a system, a device, a method for applications nowknown and later developed or a computer readable medium. These and otherunique features of the system disclosed herein will become more readilyapparent from the following description and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

So that those having ordinary skill in the art to which the disclosedsystem appertains will more readily understand how to make and use thesame, reference may be had to the following drawings.

FIG. 1 is a flow chart showing methods for application of the inventionin an embryo thawing process; and

FIG. 2 is a flow chart showing methods for application of the inventionin an embryo vitrification process.

DETAILED DESCRIPTION OF THE INVENTION

Many of the ingredients of the media of this invention have an impact,some being glucose, pyruvates and lactates, fats, vitamins, proteins andantioxidents, Amino acid concentrations are also needed in order toprovide for the synthetic requirement of cellular repair. When the timecomes to be revived or unfrozen, the specimen is then removed from theliquid nitrogen container and placed into a series of thawing media fora specific time intervals and through several steps.

The method and system of this invention will provide pre-vitrification,thawing and post thawing embryos a balance of ingredients that willprovide the embryo with those ingredients which will allow it to betterrecover from a metabolic shift and inner mitochondrial membrane damagecaused by the CP process. When the specimens are being prepared forcryopreservation the specimens are handled in several different methodsand different ingredients, depending on the selected protocols andproducts. The subject technology will increase the chances of increasedpregnancies and live child birth in the embryos cultured in the solutionas opposed to the other solutions. The subject technology will increasethe chances of stem cells recovering and becoming more useful as well agreater survival rate. Some of the combinations will include ingredientssuch as stabilizers or antifreeze proteins which may help recovery anddevelopment. The subject disclosure may include energy sources such asfructose, glucose, maltose, mannitol, sorbitol, sucrose and trehalose.Additional ingredients such as, vitamins fats, globulin and antioxidentsare included. The subject disclosure has also altered the volume ofcalcium, magnesium and phosphate to help support development.

Referring now to the drawings, FIG. 1 compares current methods ofthawing or devitrifying specimens with an embodiment of the inventionand its methods for thawing and devitrifying. It further describes thecurrent methods for the thawing and use of the specimens, compared tothe new method. In this example embryos are used as the specimen, themethods of thawing them, placing them in media solutions until they areready for transfer and then the transfer step. It will be appreciatedthat embryos, stem cells and oocytes can be treated in a similar mannerby using the resucitation solution of this invention. Current methodsmay use from 2 to 7 vials in the thawing process, and may vary in thetimes. For this example we are describing a method using 4 thawing stepsand 4 separate solutions the specimens remain in each solution. In thisexample the fourth vial is considered the last step.

In this example the frozen embryos are removed from the liquid nitrogentank 10 and placed into the first thawing solution 14. Current mediawill consist of a base media solution, which will include from 0.5-0.8mM of sucrose and 10-12 mg/ml of Human serum albumin (HSA) and maycontain 1,2 propandiol or similar cryoprotectorants. The ingredients mayvary, but are fairly consistent through the many manufacturers. Theembryo remains in the first solution 14 for approximately 5 minutes. Theembryo is then transferred to vial #2 18 for 5 minutes. Vial #2 18 maycontain a base media, from 0.2-0.5 mM of sucrose and HSA and may contain1,2 propandiol or similar cryoprotectorants. The embryo is then movedinto the Vial #3 solution 22 for approximately 10 minutes. Vial #3 22may contain a base media, from 0.1-0.2 mM of sucrose and HSA. The embryois then moved to a generic media 32, for several hours and thentransferred into the patient. In this example “generic media” refers toa media that is used for un-frozen specimens and is not differentiatedfor frozen specimens. The embryos are then moved from the generic media32 and transferred into the patient 34.

In practicing the method of this invention, the user may use the currentmethods of moving the embryos through the 1st solution 14, secondsolution 18 and the 3rd solution 22. The embryos are removed from the #3thawing solution 22 and at step 24 moved to the rejuvenating solution 60of this invention. The solution 60 is a solution which addresses theneeds of embryos that were frozen and are being thawed to be laterimplanted, and addresses the fact that the frozen embryos have differentnutritional needs than fresh embryos. In Method A the embryos are movedat 24 into the solution 60 before they are put into the last thawingsolution 30. This exposes the embryos to the enhancements of thesolution 60, due in part to the fact that most last thawing solution areonly a base media solution and HSA (human serum albumin). Therefore itis more beneficial to be in the solution 60 than in the last solution26. This example also allows the solution 60 to become the finalsolution used in the sequence of solutions and the last solution in kitscurrently provided by IVF companies.

In Method A, the embryos are held in the solution 60 for a period oftime, undefined in this example, and then may be moved in step 64 to a“generic medium” 72 for a predetermined period of time. The embryos arethen transferred to the patient 74 from the generic medium 72. Thisallows users to use any of a number of generic media that they may bemore accustomed to before they transfer the embryo. The embryo is thenmoved from 72 and transferred into the patient 74.

Alternatively in Method A, the embryos may be held in the solution 60until they are transferred into the patient 76, through step 68.Therefore this embodiment is a unique combination of steps and is usedin the thawing and recovery process of the embryos as well as used forthe growth and culturing of the embryos through to the transfer stage74.

In Method B, the embryos are placed in the Method B last solution 30,then at step 28 are moved to the rejuvenation solution 80. The move tothe solution 80, in this example, allows the user to utilize the stepsor protocols of the current method, which are steps or protocols theymay prefer, or be more accustomed to using.

After the embryos are moved to the solution 80, they may be cultured inthe solution 80 (step 84) until they are transferred into the patient96. The user may choose to move the embryos in step 88 to a genericmedium 92, for a time period, a step which they may be more accustomedto, and then the embryos can be implanted into the patient 96.

In this example the media solutions of 32, 72 and 92 may be genericmedium, may be a “transfer” medium, or may be a combination of both.Transfer medium, in this example, is a medium designed for the step oftransferring the embryo to the patient and may include additionalingredients, such as hyaluronic acid. This example demonstrates theunique nature of the rejuvenation solution, and its added specificingredients and the old Methods A and B, for the handling of the embryosand the introduction or addition of the rejuvenation solution into theolder current methods and protocols.

Referring now to FIG. 2, there is shown a similar procedure for freezingor vitrifying specimens such as embryos, stem cells or oocytes. In theprior art method, the specimens, such as embryos or oocytes to becryopreserved by freezing or vitrification are cultured in a culturingmedia 200. This occurs in an incubator environment. The embryos areselected for cryopreservation and then put through the vitrification orfreezing process in specific cryoprotectorant (CP) media solutions 202.Typically in the first vitrification step, the media will include twocyroprotectorants which will each provide 7.5% of the first step medium.Then in the second vitrification step, the cryoprotectorants will eachprovide 15% of the second step medium. After they have been thusprepared for vitrification in the are then moved to storage in a liquidnitrogen tank 204. The specimens will remain in the tank 204 untilneeded to be thawed and used.

The method of this invention includes a specially designed media in thesequence to provide the embryos with much needed components before theyenter the vitrification process. The embryos are cultured in an embryoculture media 220, before cryopreservation. The embryos are moved fromthe generic culture medium into a preparation/rejuvenation medium 225Awhich contains the combination of ingredients, described above and isdevoid of HEPES or MOPS. This special solution contains additionalingredients useful in preparing the specimen for cryopreservation. Theembryos are then moved into a second preparation/rejuvenation medium225B which does contain HEPES or MOPS. Including HEPES or MOPS, abicarbonate, allows the embryos to be handled outside the incubatorenvironment, and includingn HEPES or MOPS allows the user to prepare theembryos inside of the incubator environment for a longer period of time.The embryos are then placed in the sequential vitrification solutions230 and 231, and then stored in liquid nitrogen tank 275.

Referring now to the tables, Table 1 shows the changes made to a basicmedia formulation A. This table shows what is a generic medium solutionthat has been successful in developing embryos through to the cleavageand blastocyst stage of development. This table shows the majority ofingredients used in this proven medium. The combination of pyruvates,glucose and lactate in the rejuvenation solution will outperform theconventional media in the post thawing process. Added to the provenmedia formulations are varying amounts of fatty acids, vitamins,antioxidents and globulins.

The term “amino acid” as used herein, refers to amino acids or theirderivatives. Examples of such amino acids include L-alamine, glycine,L-alanine, L-arginine HCL, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, glycyl-glutamine, L-phenylalanine, L-histidine,L-isoleucine, L-lysine HCL, L-leucine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

In modifying the basic medium ingredients, glucose is increased ten foldfrom 0.036 to 0.18 g/L. Lactate is decreased from 1.12 to 0.56 gg/L.Pyruvate is increased from 0.022 to 0.030 g/L. The levels may varyslightly and acceptable levels of glucose may be from 0.20 to 0.40 g/L;lactate may vary from 0.20 to 1.20 g/L; and pyruvates may vary from0.005 to 0.030 g/L

The medium of this invention includes the addition of fatty acids,lipioc acid in a concentration of from 0.000004 to 0.0000012 g/L, with apreferred amount of 0.000008 g/L; and linoleic acid in a concentrationof from 0.000002 to 0.000008, with a preferred amount of 0.00005 g/L.The fatty acids may be supplemented with similar performance fattyacids.

The invention includes the addition of vitamins, alpha tocopheralacetate in a concentration of from 0.00025 to 0.00075 g/L, with apreferred embodiment of 0.0005 g/L, ascorbic acid phosphate in aconcentration of from 0.001 to 0.01, with a preferred amount of 0.0025g/L. Menadione K3 in a concentration of from 0.00005 to 0.0005, with apreferred amount of 0.0001 g/L. Vitamin K2 in a concentration of from0.00005 to 0.0005, with a preferred amount of 0.0001 g/L; globulin maybe added seperately or in a subcomponent such as HAS and globulin. Thevitamins may be supplemented with similar performance vitamins.

Table 2 compares some of the media considered in the prior art exampleof FIG. 1. Table 2 shows the more common “generic” media solutions inthe marketplace and their published concentration of the ingredientsglucose, sodium and sodium lactate. This includes those common in embryodevelopment and culturing. Table 2 shows that P1, a common culturemedium, which would be used as the generic medium of FIG. 1, has noglucose; three times the amount of sodium pyruvates; and more than fourtimes the amount of sodium lactate. In Table 2 there is shown that mostconcentrations of sodium lactate are at least two times the level of theresucitation culture medium of this invention. This embodiment of theresucitation culture medium of this invention has balanced and addedingredients to best culture and allow the embryos to revive afterthawing.

The invention includes the addition of fatty acids, lipioc acid in aconcentrations of from 0.000004 to 0.0000012 g/L, with a preferredconcentration of 0.000008 g/L, and linoleic acid in concentrations offrom 0.000002 to 0.000008 g/L, with a preferred concentration of 0.00005g/L. These fatty acids may be supplemented with similar performancefatty acids.

Since many changes and variations of the disclosed embodiment of theinvention may be made without departing from the inventive concept, itis not intended to limit the invention except as required by theappended claims.

What is claimed is:

1-5. (canceled)
 6. A method for thawing a frozen or vitrified embryospecimen which is contained in liquid nitrogen (LN), said methodcomprising: a) a first step of removing a specimen from said LN andplacing said specimen in a first solution comprising a base salinemedium and a first amount of sucrose as an energy source; b) a secondstep of removing said specimen from said first solution and placing saidspecimen in a second solution comprising a base saline medium and asecond amount of sucrose which is less than said first amount; c) athird step of removing said specimen from said second solution andplacing said specimen in a third solution comprising a base salinemedium and a third amount of sucrose which is less than said secondamount; and d) a fourth step of removing said specimen from said thirdsolution and placing said specimen in a rejuvenation medium comprisingeffective amounts of glucose, pyruvates, amino acids, vitamins K5 and C,antioxidants, and fatty acids sufficient to supply the specimen with thechemical ingredients and uptake requirements required to recover andprosper during and after the cryopreservation process; said solutionbeing operative to supply nutrients to the specimen which will replenishdepletion and damage to the specimen and its mitochondria, spindles andstructural features, such as cell walls.
 7. The method of claim 6further comprising a fifth step of removing said specimen from saidrejuvination medium and placing said specimen in a generic culturingmedium which will prepare said specimen for reimplantation. 8-11.(canceled)
 12. A method for thawing frozen or vitrified embryo specimenswhich are contained in liquid nitrogen (LN), said method comprising: a)a first step of removing specimens from said LN and placing saidspecimens in a first solution comprising a base saline medium and afirst amount of sucrose as an energy source; b) a second step ofremoving said specimens from said first solution and placing saidspecimens in a second solution comprising a base saline medium and asecond amount of sucrose which is less than said first amount; c) athird step of removing said specimens from said second solution andplacing said specimens in a third solution comprising a base salinemedium and a third amount of sucrose which is less than said secondamount; and d) a fourth step of removing said specimens from said thirdsolution and placing said specimens in a rejuvenation medium comprisingeffective amounts of glucose, pyruvates, amino acids, vitamin K5 andvitamin C, antioxidants, and fatty acids sufficient to supply thespecimens with the chemical ingredients and uptake requirements requiredto recover and prosper during and after the cryopreservation process;said solution being operative to supply nutrients to the specimens whichwill replenish depletion and damage to the specimens and theirmitochondria, spindles and structural features, such as cell walls. 13.A method for thawing frozen or vitrified embryo specimens which arecontained in liquid nitrogen (LN), said method comprising: a) a firststep of removing specimens from said LN and placing said specimens in afirst solution comprising a base saline medium and a first amount ofsucrose as an energy source; b) a second step of removing said specimensfrom said first solution and placing said specimens in a second solutioncomprising a base saline medium and a second amount of sucrose which isless than said first amount; c) a third step of removing said specimensfrom said second solution and placing said specimens in a third solutioncomprising a base saline medium and a third amount of sucrose which isless than said second amount; and d) a fourth step of removing saidspecimens from said third solution and placing said specimens in arejuvenation medium comprising effective amounts of glucose, pyruvates,amino acids, vitamin K and vitamin C, antioxidants, and fatty acidssufficient to supply the specimens with the chemical ingredients anduptake requirements required to recover and prosper during and after thecryopreservation process; said solution being operative to supplynutrients to the specimens which will replenish depletion and damage tothe specimens and their mitochondria, spindles and structural features,such as cell walls.